AF9 (MLLT3) was first identified as a fusion partner of the Mixed Lineage Leukemia (MLL) gene in acute myeloid leukemia. At the time it was noted to share sequence homology to a component of the yeast SWI/SNF complex TFG3/ANC1, suggesting a role in transcription. AF9 clearly plays a key role in specific lineage fate decisions in hematopoiesis. In addition, its high level of expression in HSCs strongly suggests a critical role in maintaining the gene expression program necessary for HSCs. The mechanistic basis for these roles of AF9 has not been established. One of the primary goals of this grant is to provide a molecular basis for the function of AF9 with special emphasis on the role of the critical N- and C-terminal domains and their relationship to the gene regulatory machinery. We propose that binding of the AF9 N-terminal YEATS domain to histone H3 as well as binding of the AF9 C-terminal domain to AF4 are essential for normal erythrocyte and megakaryocyte differentiation. In addition, we propose that the interaction of MLL-AF9 with AF4 is essential for neoplastic transformation. The experiments that are proposed in this grant application are organized into two specific aims. Aim 1 focuses on the N-terminal YEATS domain and Aim 2 focuses on the AF9 C-terminal domain. Aim 1: Structure and function of the AF9 YEATS domain. Our Preliminary Data shows that the AF9 YEATS domain binds specifically to a region of histone H3. We are proposing three Sub-Aims to probe the role of this interaction in AF9 function. First, we will solve the structure of the AF9 YEATS domain bound to an H3 peptide. Based on the structure, we will develop specific point mutations to disrupt this interaction which will be introduced into full-length AF9 for functional testing. Second, we will examine the role of this interaction in the regulation of specific genes encoding hematopoietic transcription factors. Third, we will examine the role of the YEATS domain - H3 interaction in AF9's role in driving erythrocyte/megakaryocyte development. Aim 2: Structure and function of the AF9 C-terminal domain - AF4 complex. We are proposing 6 Sub-Aims to examine the role of the AF9-AF4 interaction in AF9 and MLL-AF9 function. First, we will determine a structure of the AF9 C-terminal domain - AF4 complex and use this to develop mutations to disrupt the interaction. These mutations will be used to test the role of this interaction in normal AF9 function, including effects on composition of the AF9 complex, the regulation of specific genes encoding hematopoietic transcription factors, and erythrocyte/megakaryocyte development. As this is the domain fused to MLL in MLL-AF9 fusions, these mutations will also be used to test effects on regulation of HOXA9 and MEIS1 by MLL-AF9 and MLL-AF9 leukemogenesis.